The latest Zeiss confocal microscope is configured on an inverted microscope equipped with 4 Zeiss objectives: EC PlnN 10X/1.3 na, 1.11um resolution AIR; LD LCI PlnApo 25X/0.8 na, 0.42um resolution WATER, OIL, GLYCEROL; ECPlnN 40X/1.3 na, 0.26um resolution OIL; Pln Apo 63X/1.4na, 0.24um resolution OIL. The confocal is equipped with three lasers: a 488nm argon laser, a 561nm diode laser and a 633 HeNe laser and is capable of up to 10 dye simultaneous detection over the entire wavelength range (in 3nm intervals). The microscope is operated directly by a TFT touch display or via the accompanying ZEN software, which is used for image acquisition and analysis. Transmitted halogen light and UV mercury epifluorescence are filtered with GFP, RFP and DIC filter sets.
The Beckman Coulter FC500 flow cytometer is an advanced cytometer equipped with two laser lines: 488nm and 633nm. The cytometer is capable of up to five color simultaneous detection:
- FL1: 515nm- 535nm (example: FITC, GFP)
- FL2: 567nm-583nm (example: PE)
- FL3: 590nm-615nm (example: ECD)
- FL4: 660nm-690nm (example: PE-Cy5, PE-Cy5.5, PerCP, APC)
- FL5: 765nm-825nm (example: PE-Cy7)
The Olympus FV1000 2-photon confocal microscope is equipped with tungsten and halogen visible light sources and four lasers: argon 458nm, 488nm, 515nm; HeNe 543 nm; red diode 635; and a Mai Tai DeepSee titanium-sapphire 690-1040nm. The microscope has two cube filter sets allowing for distinct spectral configurations: Cube Set 1: 460-500, 520-560, 575-625, 650-700 and Cube Set 2: 425-465, 500-550, 575-625, 650-700.
Five objectives are on the microscope:
- 10X UPlanSApo, 0.4na AIR
- 20X UPlanSApo, 0.75na AIR
- 25X XLPlanN, 1.05na WATER
- 40X UPlanFlN, 1.3na, OIL
- 60C PlanApoN, 1.42 na, OIL
A temperature and humidity control chamber is also available.
The BD Biosciences FACSAria III cell sorter, BD’s most advanced cell sorter, is equipped with 405nm, 488nm, 561nm and 633nm lasers and is capable of up to nine color simultaneous detection. Multiple sorting options are available; populations can be sorted in up to four tubes, which can also be temperature-controlled by use of an available water-bath-heated sorting block. Populations or single cells may also be sorted directly onto slides or into standard or customized plates. Operation of the cell sorter is performed only by the core manager and must be arranged in advance by appointment.
Cell density should be 10-20 x 106 cells/ml and filtered through a 35-40µm filter. The amount of serum in final suspension buffer must be limited to < 2%, as high serum concentrations can cause distortion of light scatter signals. The healthier the cells, the better they will hold up after the sorting process. Investigators should also provide a collection medium that is appropriate for the applications (ex: DMEM +/- Serum) to be added to the collection tubes so that cells remain as healthy as possible after the sort.
Sorting is a complex process and efficiency and quality of the sort is determined by a number of different factors. Therefore, it is strongly recommended that all sorting projects be discussed with the core manager well in advance to ensure that the desired results are attainable. Even still, users should understand that different cell types sort differently, so they should expect that multiple sorts may be necessary to optimize conditions to the individual experiment. In advance, discuss what type of cells are to be sorted, what fluorochromes will be used (users should test or know the flurochrome and antibody combination well); what percentage of the desired cells are from the original population; and if sorting needs to be done into tubes or into a plate or slides.
The FACSAria III cell sorter is not affiliated with the CTSI resource cores. The IU School of Medicine—South Bend Imaging and Flow Cytometry Core facility operates the sorter, but this instrument does not carry CTSI designation and therefore is not available for CTSI core specific funding initiatives.